Production of Alkenes by Combined Enzymatic Conversion of 3-Hydroxyalkanoic Acids

ABSTRACT

The present invention relates to a method for generating alkenes through a biological process. More specifically, the invention relates to a method for producing alkenes (for example propylene, ethylene, 1-butylene, isobutylene or isoamylene) from molecules of the 3-hydroxyalkanoate type.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 13/880,042, filed on Jul.6, 2013, which is the National Phase under 35 U.S.C. §371 of PCTInternational Application No. PCT/EP2011/068174 which has aninternational filing date of Oct. 18, 2011, which claims priority toEuropean Patent Application EP10188001 filed on Oct. 19, 2010. Theentire contents of these applications listed above are herebyincorporated by reference in their entirety.

The present invention relates to a method for generating alkenes througha biological process. More specifically, the invention relates to amethod for producing alkenes (for example propylene, ethylene,1-butylene, isobutylene or isoamylene) from molecules of the3-hydroxyalkanoate type.

A large number of chemical compounds are currently derived frompetrochemicals. Alkenes (such as ethylene, propylene, the differentbutenes, or else the pentenes, for example) are used in the plasticsindustry, for example for producing polypropylene or polyethylene, andin other areas of the chemical industry and that of fuels.

Ethylene, the simplest alkene, lies at the heart of industrial organicchemistry: it is the most widely produced organic compound in the world.It is used in particular to produce polyethylene, a major plastic.Ethylene can also be converted to many industrially useful products byreaction (of oxidation, of halogenation).

Propylene holds a similarly important role: its polymerization resultsin a plastic material, polypropylene. The technical properties of thisproduct in terms of resistance, density, solidity, deformability, andtransparency are unequalled. The worldwide production of polypropylenehas grown continuously since its invention in 1954.

Butylene exists in four forms, one of which, isobutylene, enters intothe composition of methyl-tert-butyl-ether (MTBE), an anti-knockadditive for automobile fuel. Isobutylene can also be used to produceisooctene, which in turn can be reduced to isooctane(2,2,4-trimethylpentane); the very high octane rating of isooctane makesit the best fuel for so-called “gasoline” engines.

Amylene, hexene and heptene exist in many forms according to theposition and configuration of the double bond. These products have realindustrial applications but are less important than ethylene, propyleneor butenes.

All these alkenes are currently produced by catalytic cracking ofpetroleum products (or by a derivative of the Fisher-Tropsch process inthe case of hexene, from coal or gas). Their cost is therefore naturallyindexed to the price of oil. Moreover, catalytic cracking is sometimesassociated with considerable technical difficulties which increaseprocess complexity and production costs.

Independently of the above considerations, the bioproduction of plastics(“bioplastics”) is a thriving field. This boom is driven by economicconcerns linked to the price of oil, and by environmental considerationsthat are both global (carbon-neutral products) and local (wastemanagement).

The main family of bioplastics is that of the polyhydroxyalkanoates(PHA). These are polymers obtained by condensation of moleculescomprising both an acid group and an alcohol group. Condensation takesplace by esterification of the acid on the alcohol of the followingmonomer. This ester bond is not as stable as the direct carbon-carbonbond present in the polymers of conventional plastics, which explainswhy PHAs have a biodegradability of a few weeks to a few months.

The PHA family includes in particular poly-3-hydroxybutyrate (PHB), apolymer of 3-hydroxybutyrate, and polyhydroxybutyrate-valerate (PHBV),an alternating polymer of 3-hydroxybutyrate and 3-hydroxyvalerate.

PHB is naturally produced by some strains of bacteria such asAlcaligenes eutrophus and Bacillus megaterium. Laboratory bacteria, likeE. coli, having integrated synthetic pathways leading to PHB or to PHAsin general, have been constructed. The compound or its polymer can, incertain laboratory conditions, account for up to 80% of the bacterialmass (Wong M S et al., Biotech. Bioeng. 99 (2008), 919-928).Industrial-scale production of PHB was attempted in the 1980s, but thecosts of producing the compound by fermentation were considered too highat the time. Projects involving the direct production of these compoundsin genetically modified plants (having integrated the key enzymes of thePHB synthetic pathway present in producer bacteria) are in progress andmight entail lower operating costs.

The biological production of alkanes or other hydrocarbon molecules thatcan be used as fuels or as precursors of synthetic resins is called forin the context of a sustainable industrial operation in harmony withgeochemical cycles. The first generation of biofuels consisted in thefermentative production of ethanol, as fermentation and distillationprocesses already existed in the food processing industry. Theproduction of second generation biofuels is in an exploratory phase,encompassing in particular the production of long chain alcohols(butanol and pentanol), terpenes, linear alkanes and fatty acids. Tworecent reviews provide a general overview of research in this field:Ladygina N et al., Process Biochemistry, 2006, 41:1001; and Wackett L P,Current Opinions in Chemical Biology, 2008, 21:187.

In the alkene chemical family, isoprene (2-methyl-1,3-butadiene) is theterpene motif which, through polymerization, leads to rubber. Otherterpenes might be developed, by chemical, biological or mixed pathways,as usable products such as biofuels or to manufacture plastics. Therecent literature shows that the mevalonate pathway (a key intermediatein steroid biosynthesis in many organisms) might be used in order toefficiently produce products from the terpene family at industrialyields (Withers S T et al., Appl. Environ. Microbiol., 2007, 73:6277).

The production of alkenes, in particular terminal alkenes, [ethylenemono- or di-substituted at position 2: H₂C═C(R¹)(R²)] has apparentlybeen less extensively investigated. The conversion of isovalerate toisobutylene by the yeast Rhodotorula minuta has been described (Fujii T.et al., Appl. Environ. Microbiol., 1988, 54:583), but the efficiency ofthis reaction, characterized by a very low value of the turnover number(k_(cat) is 1×10⁻⁵ sec⁻¹), is far from permitting an industrialapplication. The reaction mechanism was elucidated by Fukuda H et al.(BBRC, 1994, 201(2):516) and involves a cytochrome P450 enzyme whichdecarboxylates isovalerate by reduction of an oxoferryl group Fe^(v)═O.At no point does the reaction involve hydroxylation of isovalerate.Isovalerate is also an intermediate in leucine catabolism. Large-scalebiosynthesis of isobutylene by this pathway seems highly unfavorable,since it would require the synthesis and degradation of one molecule ofleucine to form one molecule of isobutylene. Also, the enzyme catalyzingthe reaction uses heme as cofactor, poorly lending itself to recombinantexpression in bacteria and to improvement of enzyme parameters. For allthese reasons, it appears very unlikely that this pathway of the priorart can serve as a basis for industrial exploitation. Othermicroorganisms have been described as being marginally capable ofnaturally producing isobutylene from isovalerate; the yields obtainedare even lower than those obtained with Rhodotorula minuta (Fukuda H. etal, Agric. Biol. Chem., 1984, 48:1679).

The same studies have also described the natural production ofpropylene: many microorganisms are capable of producing propylene, onceagain with an extremely low yield. The production of ethylene by plantshas long been known (Meigh et al, 1960, Nature, 186:902). According tothe metabolic pathway elucidated, methionine is the precursor ofethylene (Adams and Yang, PNAS, 1979, 76:170). Conversion of2-oxoglutarate has also been described (Ladygina N et al., ProcessBiochemistry 2006, 41:1001). Since the production of a two-carbonmolecule of ethylene consumes a four- or five-carbon molecule precursor,these pathways appear materially and energetically unfavorable for theirindustrial application.

Thus, there is a need for efficient methods for producing alkenes suchas ethylene, propylene, 1-butylene, isobutylene, 1-amylene orisoamylene.

WO2010/001078 describes a process for producing alkenes by enzymaticconversion of 3-hydroxyalkanoic acids with an enzyme having the activityof a decarboxylase. Such a method is advantageous because it helps toavoid the use of petroleum products, to lower the costs of producingplastics and fuels and can have a considerable global environmentalimpact by allowing carbon to be stored in solid form. Although themethod described in WO 2010/001078 allows to produce alkenes byenzymatically converting 3-hydroxyalkanoates, there is still a need forimprovements, in particular as regards efficiency of the process so asto make it suitable for industrial purposes. The present applicationaddresses this need.

The present invention describes a method for producing alkene compoundsstarting from a 3-hydroxyalkanoate through a biological process, inparticular an enzymatic process, in which two types of enzymes arecombined in order to increase the efficiency of the production rate.More specifically, the present invention relates to a method forproducing an alkene, characterized in that it comprises the conversionof a 3-hydroxyalkanoate into said alkene by

-   -   (i) a first enzyme having an activity of converting the        3-hydroxyalkanoate into the corresponding        3-phosphonoxyalkanoate; and    -   (ii) a second enzyme being different from the first enzyme and        having an activity of converting said 3-phosphonoxyalkanoate        into said alkene.

The present invention also relates to the use of at least two enzymes,wherein one enzyme is selected from (i) as specified above and the otherenzyme is selected from (ii) as specified above or of a microorganismproducing said combination of enzymes, for producing an alkene compoundfrom a 3-hydroxyalkanoate.

The present invention also relates to organisms, preferablymicroorganisms, which produce at least two enzymes, wherein one enzymeis selected from (i) as specified above and the other enzyme is selectedfrom (ii) as specified above.

“3-hydroxyalkanoate”, as used herein, denotes a molecule responding tothe following general formula:

C_(n+1)H_(2n+2)O₃,

with 1<n<7, and comprising 3-hydroxypropionate as a common motif (FIG.1), and optionally one or two alkyl substitutions on carbon 3. Saidalkyl residues or groups can be linear or branched. As used herein, theterms “alkoyl” and “alkyl” have the same meaning and areinterchangeable. Likewise, the terms “residue” and “group” have the samemeaning and are interchangeable. Methyl, ethyl, propyl, isopropyl,butyl, isobutyl groups are examples of said alkyl groups. Carbon 3becomes a chiral center if the two alkyl substitutions are different.The present definition encompasses the two chiral forms, even if one ofthe two forms, for example the R form, is the main form producednaturally. Examples of 3-hydroxyalkanoates are presented in FIG. 3.Optionally, alkyl substituents can be added on carbon 2, which then mayalso become chiral (if the two substituents are different). Equally, theconfigurations of the 3-hydroxyalkanoate substrates in the presentinvention encompass all the stereoisomers. In a preferred embodiment,the 3-hydroxyalkanoates correspond either to 3-hydroxypropionate or tovariants or derivatives of 3-hydroxypropionate in which one of the twoor the two hydrogen atoms carried on carbon 3 are substituted by a motifcomposed solely of carbon and hydrogen atoms, the number of carbon atomsof said substituents ranging from 1 to 5, preferably from 1 to 3, suchas methyl, ethyl, propyl, isopropyl, butyl or isobutyl. The suffix“oate”, as used herein, can interchangeably denote either thecarboxylate ion (COO—) or carboxylic acid (COON). It is not used todenote an ester. In a particular embodiment, the 3-hydroxyalkanoates arerepresented by the following formula: HO—CO—CH₂—C(R¹)(R²)—OH orO⁻—CO—CH₂—C(R¹)(R²)—OH.

The term “3-phosphonoxyalkanoate” denotes a molecule which responds tothe following general formula:

C_(n+1)H_(2n+3)O₆P,

with 1<n<7, and comprising 3-phosphonoxypropionate as a common motif,and optionally one or two alkyl substitutions on carbon 3.

The term “alkene”, as used herein, denotes molecules composed solely ofcarbon and hydrogen, containing one carbon-carbon double bond and havingthe chemical formula of a mono-unsaturated hydrocarbon, C_(n)H_(2n),where n equals at least two. Preferably, n equals at least 3, 4, 5 or 6.Most preferably n is at most 6. Thus, generally, the term “alkene”refers to a molecule responding to the formula C_(n)H_(2n), with 1<n<7.

In a preferred embodiment alkenes are represented by the structuralformula H₂C═C(R¹)(R²) wherein R¹ and R² are selected, independently,from the group consisting of a hydrogen atom and a linear or branchedalkyl radical, so that the total number of carbon atoms in the alkenemolecule is at most 6.

Preferred examples of alkene compounds according to the invention are inparticular ethylene, propylene, isobutylene, and isoamylene (FIG. 4), orelse 1-butylene and 1-amylene.

“Carbon source”, as used herein, denotes any carbon compound that can beused as substrate for the organisms according to the invention. Saidterm includes glucose or any other hexose, xylose or any other pentose,polyols such as glycerol, sorbitol or mannitol, or else polymers such asstarch, cellulose or hemicellulose, or else poly-3-hydroxyalkanoateslike poly-3-hydroxybutyrate. It may be any substrate allowing the growthof microorganisms, such as formate for example. It may also be CO₂ inthe case where the organisms are capable of carrying out photosynthesis.

“Recombinant”, as used herein, denotes the artificial geneticmodification of an organism, either by addition, removal, ormodification of a chromosomal or extra-chromosomal gene or regulatorymotif such as a promoter, or by fusion of organisms, or by addition of avector of any type, for example plasmidic. The term “recombinantexpression” denotes the production of a protein involving a geneticmodification, preferably in order to produce a protein of exogenous orheterologous origin with respect to its host, that is, which does notnaturally occur in the production host, or in order to produce amodified or mutated endogenous protein.

“Overexpression” or “overexpressing”, as used herein, denotes therecombinant expression of a protein in a host organism, preferablyoriginating from an organism different from the one in which it isexpressed, increased by at least 10% and preferably by 20%, 50%, 100%,500% and possibly more as compared to the natural expression of saidprotein occurring in said host organism. This definition alsoencompasses the case where there is no natural expression of saidprotein.

A “co-substrate” is a compound or molecule added to the enzymaticreaction, so as to improve certain parameters thereof, and above all theactivity thereof, said product and the principal substrate beingconsumed in equal amounts. The co-substrate must therefore be added tothe reaction at a concentration comparable to that of the principalsubstrate. Depending on the enzyme, the presence of a co-substrate maybe required for the enzymatic reaction.

A “cofactor” is a product added to the enzymatic reaction, so as toimprove certain parameters thereof and above all to improve the activitythereof, said product not being consumed during the reaction, andtherefore needing only to be added at a low concentration, proportionalto the amount of enzyme, said concentration therefore being referred toas “catalytic”.

A “part” of an amino acid sequence denotes a fragment comprising atleast 10, preferably at least 20, 30, 40 or 50 consecutive amino acidresidues of said sequence.

“Homology”, as used herein, denotes the existence of a similaritybetween two sequences as measured by the percent identity between saidtwo sequences. In a preferred embodiment the term “homology” meanssequence identity.

Chemical compounds are often known by several names, official or common.Herein, the common names of the molecules are preferred. Thus:

-   -   “ethylene” is used to denote ethene    -   “propylene” is used to denote propene    -   “butylene” is used to denote butene    -   “isobutylene” is used to denote 2-methylpropene or isobutene    -   “amylene” is used to denote pentene    -   “isoamylene” is used to denote 2-methyl-but-1-ene or isopentene    -   “propionate” is used to denote propanoic acid or the propanoate        ion    -   “butyrate” is used to denote butanoic acid or the butanoate ion    -   “valerate” is used to denote pentanoic acid or the pentanoate        ion.

The present invention describes a method for producing alkene compoundsstarting from a 3-hydroxyalkanoate through a biological process, inparticular an enzymatic process, in which two types of enzymes arecombined in order to increase the efficiency of the production rate.More specifically, the present invention relates to a method forproducing an alkene, characterized in that it comprises the conversionof a 3-hydroxyalkanoate into said alkene by

-   -   (i) a first enzyme having an activity of converting the        3-hydroxyalkanoate into the corresponding        3-phosphonoxyalkanoate; and    -   (ii) a second enzyme being different from the first enzyme and        having an activity of converting said 3-phosphonoxyalkanoate        into said alkene.

As mentioned above, WO 2010/001078 describes a process for producingalkenes by enzymatic conversion of 3-hydroxyalkanoic acids with anenzyme having the activity of a decarboxylase. It has been described inWO 2010/001078 that generally the conversion of a 3-hydroxyalkanoateinto an alkene by an enzyme having a decarboxylase activity, e.g. amevalonate diphosphate (MDP) decarboxylase (E.C. 4.1.1.33) takes placeby the conversion of the 3-hydroxyalkanoate into the corresponding3-phosphonoxyalkanoate which is then decarboxylated to lead to thecorresponding alkene. The generic reaction carried out by MDPdecarboxylase using various 3-hydroxyalkanoates is depicted in FIG. 2B.

It has now been found that different decarboxylases, in particularmevalonate diphosphate decarboxylases, catalyze the two above mentionedsteps with different efficiencies, i.e. that some decarboxylasescatalyze the first step with a higher efficiency than otherdecarboxylases and that some decarboxylases show a preference for thesecond step, i.e. the decarboxylation step, and that therefore theefficiency of the conversion of the 3-hydroxyalkanoate into the alkeneas described in WO 2010/001078 can be significantly increased bycombining corresponding enzymes. Thus, the present invention inparticular relates to a method for achieving a higher efficiency in theenzymatic production of alkenes from 3-hydroxyalkanoates, i.e. a methodfor improving the efficiency of such an enzymatic production.

The term “an enzyme having an activity of converting the3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate” meansan enzyme which can phosphorylate a 3-hydroxyalkanoate into thecorresponding 3-phosphonoxyalkanoate. The phosphate group comespreferably from an ATP molecule.

This activity can, e.g., be measured as described in the attachedExamples, in particular Example 5. One possibility is thus to incubatethe respective enzyme with the 3-hydroxyalkanoate and ATP and to measurethe production of ADP (which reflects the production of thecorresponding 3-phosphonoxyalkanoate). Assays for measuring theproduction of ADP are known to the person skilled in the art. One ofthese methods is the pyruvate kinase/lactate dehydrogenase assaydescribed in Example 5. In this case the assay measures the rate of NADHabsorbance decrease at 340 nm which is proportional to the ADP quantity.In a preferred embodiment the term “an enzyme having an activity ofconverting the 3-hydroxyalkanoate into the corresponding3-phosphonoxyalkanoate” means an enzyme which can convert3-hydroxyisovalerate and ATP into 3-phosphonoxyisovalerate and ADP. Evenmore preferably such an enzyme can catalyze the reaction of convertingthe 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate,preferably the reaction of converting 3-hydroxyisovalerate and ATP into3-phosphonoxyisovalerate and ADP, with a K_(M) of 10 mM or lower, e.g.with a K_(M) of 5 mM or lower, preferably of 1 mM or lower and even morepreferably of 0.1 mM or lower. In a particularly preferred embodimentsuch an enzyme can catalyze the reaction of converting the3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate,preferably the reaction of converting 3-hydroxyisovalerate and ATP into3-phosphonoxyisovalerate and ADP, with a k_(cat) of at least 0.2 s⁻¹,preferably with a k_(cat) of at least 0.5 s⁻¹, particularly preferredwith a k_(cat) of at least 1.0 s⁻¹, more preferred of at least 2.0 s⁻¹and even more preferred with a k_(cat) of at least 5.0 s⁻¹.

In a particularly preferred embodiment the capacity to convert3-hydroxyisovalerate and ATP into 3-phosphonoxyisovalerate and ADP ismeasured in an assay as described in Example 5.

The term “an enzyme having an activity of converting said3-phosphonoxyalkanoate into said alkene” means an enzyme which cancatalyze a reaction by which there is a decarboxylation anddephosporylation of the 3-phosphonoxyalkanoate thereby leading to thecorresponding alkene.

This activity can, e.g., be measured as described in the appendedExamples, in particular in Example 8. One possibility is thus toincubate the respective enzyme with the correspondingphosphonoxyalkanoate under conditions which in principle allow thedecarboxylation and the dephosphorylation and to detect the productionof the corresponding alkene, e.g. by gas chromatography. In a preferredembodiment the term “an enzyme having an activity of converting said3-phosphonoxyalkanoate into said alkene” means an enzyme which canconvert 3-phosphonoxyisovalerate into isobutene, preferably under theconditions described in Example 8. Even more preferably such an enzymecan catalyze the reaction of converting the 3-phosphonoxyalkanoate intothe corresponding alkene (via decarboxylation and dephosphorylation)with a K_(M) of 100 mM or lower, e.g. with a K_(M) of 75 mM or lower, orwith a K_(M) of 50 mM or lower, preferably of 10 mM or lower or 5 mM orlower or 1 mM or lower, and even more preferably of 0.1 mM or lower. Ina particularly preferred embodiment such an enzyme can catalyze thereaction of converting the 3-phosphonoxyalkanoate into the correspondingalkene, preferably the reaction of converting 3-phosphonoxyisovalerateinto isobutene, with a k_(cat) of at least 10⁻⁶ s⁻¹, preferably with ak_(cat) of at least 10⁻⁴ s⁻¹, e.g. with a k_(cat) of at least 10⁻³ s⁻¹or with a k_(cat) of at least 10's⁻¹, such as with a k_(cat) of at least10⁻¹ s⁻¹, for example with a k_(cat) of at least 0.2 s⁻¹, preferablywith a k_(cat) of at least 0.5 s⁻¹, particularly preferred with ak_(cat) of at least 1.0 s⁻¹, more preferred of at least 2.0 s⁻¹ and evenmore preferred with a k_(cat) of at least 5.0 s⁻¹.

In a particularly preferred embodiment the capacity to convert3-phosphonoxyisovalerate into isobutene is measured in an assay asdescribed in Example 8.

In one preferred embodiment an enzyme mentioned in (i) and (ii), above,is an enzyme which is considered by NCBl or an equivalent engine ashaving a COG3407 domain.

In a preferred embodiment of the method according to the invention thefirst enzyme (i) having an activity of converting the 3-hydroxyalkanoateinto the corresponding 3-phosphonoxyalkanoate is selected from the groupconsisting of

-   -   (A) a protein comprising the amino acid sequence as shown in SEQ        ID NO: 1 or a protein comprising an amino acid sequence which is        at least 15% identical to the amino acid sequence shown in SEQ        ID NO: 1 and showing an activity of converting the        3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate        which is at least as high as the corresponding activity of the        protein having the amino acid sequence shown in SEQ ID NO: 1;    -   (B) a protein comprising the amino acid sequence as shown in SEQ        ID NO: 2 or a protein comprising an amino acid sequence which is        at least 15% identical to the amino acid sequence shown in SEQ        ID NO: 2 and showing an activity of converting the        3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate        which is at least as high as the corresponding activity of the        protein having the amino acid sequence shown in SEQ ID NO: 2;    -   (C) a protein comprising the amino acid sequence as shown in SEQ        ID NO: 3 or a protein comprising an amino acid sequence which is        at least 15% identical to the amino acid sequence shown in SEQ        ID NO: 3 and showing an activity of converting the        3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate        which is at least as high as the corresponding activity of the        protein having the amino acid sequence shown in SEQ ID NO: 3;        and    -   (D) a protein comprising the amino acid sequence as shown in SEQ        ID NO: 4 or a protein comprising an amino acid sequence which is        at least 15% identical to the amino acid sequence shown in SEQ        ID NO: 4 and showing an activity of converting the        3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate        which is at least as high as the corresponding activity of the        protein having the amino acid sequence shown in SEQ ID NO: 4.

SEQ ID NO: 1 shows the amino acid sequence of an enzyme from Picrophilustorridus DSM 9790 (GenBank accession number AAT43941; Swissprot/TrEMBLaccession number Q6KZB1).

SEQ ID NO: 2 shows the amino acid sequence of an enzyme fromThermoplasma acidophilum (GenBank accession number CAC12426;Swissprot/TrEMBL accession number Q9HIN1).

SEQ ID NO: 3 shows the amino acid sequence of an enzyme fromThermoplasma volcanium (GenBank accession number BAB59465;Swissprot/TrEMBL accession number Q97BY2).

SEQ ID NO: 4 shows the amino acid sequence of an enzyme from Ferroplasmaacidarmanus fer1 (GenBank accession number ZP_(—)05571615).

In a further preferred embodiment of the method according to theinvention the second enzyme (ii) having an activity of converting said3-phosphonoxyalkanoate into said alkene is selected from the groupconsisting of

-   -   (a) a protein comprising the amino acid sequence as shown in SEQ        ID NO: 5 or a protein comprising an amino acid sequence which is        at least 15% identical to the amino acid sequence shown in SEQ        ID NO: 5 and showing an activity of converting said        3-phosphonoxyalkanoate into said alkene which is at least as        high as the corresponding activity of the protein having the        amino acid sequence shown in SEQ ID NO: 5;    -   (b) a protein comprising the amino acid sequence as shown in SEQ        ID NO: 6 or a protein comprising an amino acid sequence which is        at least 15% identical to the amino acid sequence shown in SEQ        ID NO: 6 and showing an activity of converting said        3-phosphonoxyalkanoate into said alkene which is at least as        high as the corresponding activity of the protein having the        amino acid sequence shown in SEQ ID NO: 6;    -   (c) a protein comprising the amino acid sequence as shown in SEQ        ID NO: 7 or a protein comprising an amino acid sequence which is        at least 15% identical to the amino acid sequence shown in SEQ        ID NO: 7 and showing an activity of converting said        3-phosphonoxyalkanoate into said alkene which is at least as        high as the corresponding activity of the protein having the        amino acid sequence shown in SEQ ID NO: 7;    -   (d) a protein comprising the amino acid sequence as shown in SEQ        ID NO: 8 or a protein comprising an amino acid sequence which is        at least 15% identical to the amino acid sequence shown in SEQ        ID NO: 8 and showing an activity of converting said        3-phosphonoxyalkanoate into said alkene which is at least as        high as the corresponding activity of the protein having the        amino acid sequence shown in SEQ ID NO: 8;    -   (e) a protein comprising the amino acid sequence as shown in SEQ        ID NO: 9 or a protein comprising an amino acid sequence which is        at least 15% identical to the amino acid sequence shown in SEQ        ID NO: 9 and showing an activity of converting said        3-phosphonoxyalkanoate into said alkene which is at least as        high as the corresponding activity of the protein having the        amino acid sequence shown in SEQ ID NO: 9;    -   (f) a protein comprising the amino acid sequence as shown in SEQ        ID NO: 10 or a protein comprising an amino acid sequence which        is at least 15% identical to the amino acid sequence shown in        SEQ ID NO: 10 and showing an activity of converting said        3-phosphonoxyalkanoate into said alkene which is at least as        high as the corresponding activity of the protein having the        amino acid sequence shown in SEQ ID NO: 10;    -   (g) a protein comprising the amino acid sequence as shown in SEQ        ID NO: 11 or a protein comprising an amino acid sequence which        is at least 15% identical to the amino acid sequence shown in        SEQ ID NO: 11 and showing an activity of converting said        3-phosphonoxyalkanoate into said alkene which is at least as        high as the corresponding activity of the protein having the        amino acid sequence shown in SEQ ID NO: 11;    -   (h) a protein comprising the amino acid sequence as shown in SEQ        ID NO: 12 or a protein comprising an amino acid sequence which        is at least 15% identical to the amino acid sequence shown in        SEQ ID NO: 12 and showing an activity of converting said        3-phosphonoxyalkanoate into said alkene which is at least as        high as the corresponding activity of the protein having the        amino acid sequence shown in SEQ ID NO: 12;    -   (i) a protein comprising the amino acid sequence as shown in SEQ        ID NO: 13 or a protein comprising an amino acid sequence which        is at least 15% identical to the amino acid sequence shown in        SEQ ID NO: 13 and showing an activity of converting said        3-phosphonoxyalkanoate into said alkene which is at least as        high as the corresponding activity of the protein having the        amino acid sequence shown in SEQ ID NO: 13;    -   (j) a protein comprising the amino acid sequence as shown in SEQ        ID NO: 14 or a protein comprising an amino acid sequence which        is at least 15% identical to the amino acid sequence shown in        SEQ ID NO: 14 and showing an activity of converting said        3-phosphonoxyalkanoate into said alkene which is at least as        high as the corresponding activity of the protein having the        amino acid sequence shown in SEQ ID NO: 14; and    -   (k) a protein comprising the amino acid sequence as shown in SEQ        ID NO: 15 or a protein comprising an amino acid sequence which        is at least 15% identical to the amino acid sequence shown in        SEQ ID NO: 15 and showing an activity of converting said        3-phosphonoxyalkanoate into said alkene which is at least as        high as the corresponding activity of the protein having the        amino acid sequence shown in SEQ ID NO: 15.

SEQ ID NO: 5 shows the amino acid sequence of an enzyme cloned fromStreptococcus gordonii. SEQ ID NO: 6 shows the amino acid sequence of anenzyme from Streptococcus gordonii str. Challis substr. CH1 (GenBankaccession number AAT43941; Swissprot/TrEMBL accession number A8UU9). SEQID NO: 7 shows the amino acid sequence of an enzyme from Streptococcusinfantarius subsp infantarius ATCC BAA-102 (GenBank accession numberEDT48420.1; Swissprot/TrEMBL accession number B1SCG0). SEQ ID NO: 8shows the amino acid sequence of an enzyme from Homo sapiens (GenBankaccession number AAC50440.1; Swissprot/TrEMBL accession numberP53602.1). SEQ ID NO: 9 shows the amino acid sequence of an enzyme fromLactobacillus delbrueckii (GenBank accession number CAI97800.1;Swissprot/TrEMBL accession number Q1GAB2). SEQ ID NO: 10 shows the aminoacid sequence of an enzyme from Streptococcus mitis (strain B6) (GenBankaccession number CBJ22986.1). SEQ ID NO: 11 shows the amino acidsequence of an enzyme from Streptococcus gallolyticus UCN34 (GenBankaccession number CBI13757.1). SEQ ID NO: 12 shows the amino acidsequence of an enzyme from Streptococcus sanguinis SK36 (GenBankaccession number ABN43791.1). SEQ ID NO: 13 shows the amino acidsequence of an enzyme from Streptococcus sp. M143 (GenBank accessionnumber EFA24040.1). SEQ ID NO: 14 shows the amino acid sequence of anenzyme from Streptococcus suis 89/1591 (GenBank accession numberEEF63672.1). SEQ ID NO: 15 shows the amino acid sequence of an enzymefrom Streptococcus salivarius SK126 (GenBank accession number EEK09252).

In a preferred embodiment of the method according to the invention thefirst enzyme (i) is as defined in (A) above and the second enzyme (ii)is as defined in (a) or (b) mentioned above, even more preferably thesecond enzyme is as defined in (f), (g), (h), (i), (j) or (k) mentionedabove. As illustrated in the examples, the combination of these enzymesis particularly efficient at producing alkene compounds according to thepresent invention.

In another preferred embodiment of the method according to the inventionthe second enzyme (ii) having an activity of converting said3-phosphonoxyalkanoate into said alkene is selected from any one of theproteins listed in the following Table or from a protein comprising anamino acid sequence which is at least 15% identical to the amino acidsequence of such a protein and showing an activity of converting said3-phosphonoxyalkanoate into said alkene which is at least as high as thecorresponding activity of said protein.

TABLE 1 Organism Ref sequence GenBank Methanosarcina mazei AAM31457.1Methanocaldococcus jannaschii AAB98390.1 Staphylococcus saprophyticusBAE19266.1 Streptococcus agalactiae EA073731.1 Enterococcus faecalisAA080711.1 Flavobacterium johnsoniae ABQ04421.1 Bdellovibriobacteriovorus CAE79505.1 Chloroflexus aurantiacus A9WEU8.1 Legionellapneumophila CAH13175.1 Listeria monocytogenes EAL09343.1 Metallosphaerasedula ABP95731.1 Staphylococcus epidermidis AA003959.1 Streptococcusthermophilus AAV60266.1 Bacillus coagulans EAY45229.1 Chloroflexusaggregans EAV09355.1 Lactobacillus brevis ABJ64001.1 Lactobacillusfermentum BAG27529.1 Lactobacillus plantarum CAD64155.1 Lactobacillussalivarius ABD99494.1 Lactococcus lactis sp. lactis AAK04503.1Dichelobacter nodosus ABQ14154.1 Flavobacterium psychrophilum CAL42423.1Streptococcus pneumoniae EDT95457.1 Streptococcus pyogenes AAT86835.1Streptococcus suis ABP91444.1 Staphylococcus haemolyticus BAE05710.1Streptococcus equi ACG62435.1 Arabidopsis thaliana AAC67348.1 Borreliaafzelii ABH01961.1 Encephalitozoon cuniculi CAD25409.1 Streptomyces sp.BAB07791.1 Streptococcus agalactiae EA073731.1 Streptococcus uberisCAR41735.1 Gallus gallus XP_423130 Salmo salmar ACI34234 Natromonaspharaonis CAI48881.1 Haloarcula marismortui AAV46412.1 Haloquadratumwalsbyi CAJ51653.1

As mentioned above, not only the proteins having the specificallymentioned amino acid sequences listed in the respective SEQ ID NOs or inTable 1 can be used, but also proteins which are considered by NCBl oran equivalent engine as having a COG3407 domain and, more preferred,proteins the amino acid sequence of which shows a homology of at least15% to the specifically mentioned amino acid sequence and which have arespective enzymatic activity at least as high as the activity of aprotein having the specifically mentioned amino acid sequence. Preferredenzymes advantageously have at least x % homology, wherein x is selectedfrom the group consisting of 20, 25, 20, 35, 40, 45, 50, 55 and 60. In afurther preferred embodiment the enzyme has at least 65% sequencehomology, preferably at least 70%, more preferably at least 75%, evenmore preferably, at least 80, 85, 90, 95, 96, 97, 98 or 99% homology toone of the sequences shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14 or 15 or to one of the sequences shown in Table 1. Thepercent of sequence homology can be determined by different methods andby means of software programs known to one of skill in the art, such asfor example the CLUSTAL method or BLAST and derived software, or byusing a sequence comparison algorithm such as that described byNeedleman and Wunsch (J. Mol. Biol., 1970, 48:443) or Smith and Waterman(J. Mol. Biol., 1981, 147:195).

Such proteins showing the indicated degree of homology can, e.g., beother enzymes which occur naturally or which have been preparedsynthetically. They include in particular enzymes which can be selectedfor their ability to produce alkenes according to the invention. Thus, aselection test comprises contacting the purified enzyme, or amicroorganism producing the enzyme, with the substrate of the reactionand measuring the production of the respective compound, i.e. the3-phosphonoxyalkanoate or the alkene. Such tests are described in theexperimental section. Such selection tests can also be used to screenfor enzymes with an optimized enzymatic activity for the substrate to beconverted into the 3-phosphonoxyalkanoate or the alkene, i.e. having anoptimized activity with respect to one or more 3-hydroxyalkanoates or3-phosphonoxyalkanoates.

Such screening methods are well-known in the art and include, e.g.protein engineering techniques such as random mutagenesis, massivemutagenesis, site-directed mutagenesis, DNA shuffling, syntheticshuffling, in vivo evolution, or complete synthesis of genes andsubsequent screening for the desired enzymatic activity.

The enzyme used in the invention can thus be natural or synthetic, andproduced by chemical, biological or genetic means. It can also bechemically modified, for example in order to improve its activity,resistance, specificity, purification, or to immobilize it on a support.

It has been found that enzymes which are able to catalyze the abovedescribed reactions for converting a 3-hydroxyalkanoate into an alkenevia a 3-phospho-hydroxyalkanoate are often enzymes which can beclassified in the phylogenetic superfamily of mevalonate diphosphate(MDP) decarboxylases (enzyme nomenclature EC 4.1.1.33). MDPdecarboxylase is an enzyme involved in cholesterol biosynthesis. Saidenzyme has been isolated from a variety of organisms including animals,fungi, yeasts and some bacteria. It can also be expressed by some plants(Lalitha et al., Phytochemistry 24 (11), (1985), 2569-2571). Many genesencoding this enzyme have been cloned and sequenced. These enzymes aregenerally composed of 300 to 400 amino acids and use ATP asco-substrate, which is converted during the reaction to ADP andinorganic phosphate. The phosphate group is transferred from the ATPmolecule to the tertiary alcohol of mevalonate diphosphate, releasingADP. The reaction intermediate phosphorylated on the 3-hydroxyl groupthen undergoes elimination of the phosphate group, in the physiologicalcase releasing isopentenyl diphosphate (FIG. 2).

Accordingly, in a preferred embodiment, the enzyme defined in (i) or(ii) above, is a MDP decarboxylase. In the context of the presentinvention a MDP decarboxylase is defined as an enzyme which can at leastcatalyze the conversion of 5-diphospho-3-phosphomevalonate intoisopentenyl-5-diphosphate and CO₂ or which can at least catalyze thereaction of converting mevalonate diphosphate and ATP into5-diphospho-3-phosphomevalonate and ADP. Preferably, such an enzyme cancatalyze both reactions.

In another preferred embodiment the enzyme defined in (i) above, is anenzyme as defined in (i) (B). The sequence shown in SEQ ID NO: 2represents an enzyme identified in Thermoplasma acidophilum. In Genbankthis enzyme is classified as a mevalonate diphosphate decarboxylase.However, it is known from Chen and Poulter (Biochemistry 49 (2010),207-217) that in Th. acidophilum there exists an alternative mevalonatepathway which involves the action of a mevalonate-5-monophosphatedecarboxylase. Thus, it is possible that the enzyme represented by SEQID NO: 2 actually represents a mevalonate-5-monophosphate decarboxylase.The same may hold true for other archae bacteria. Therefore, in anotherpreferred embodiment the enzyme defined in (i) or (ii) above, is amevalonate-5-monophosphate decarboxylase. Such an enzyme is capable ofconverting mevalonate-5-monophosphate into isopentenylpyrophosphate.

In preferred embodiments of the invention:

-   -   3-hydroxypropionate is converted via 3-phosphonoxypropionate        into ethylene; or    -   3-hydroxybutyrate is converted via 3-phosphonoxybutyrate into        propylene; or    -   3-hydroxyvalerate is converted via 3-phosphonoxyvalerate into        1-butylene; or    -   3-hydroxy-3-methylbutyrate (or 3-hydroxyisovalerate) is        converted via 3-phosphonoxy-3-methylbutyrate        (3-phosphonoxyisovalerate) into isobutylene; or    -   3-hydroxy-3-methylvalerate is converted via        3-phosphonoxy-3-methylvalerate into isoamylene.

The method according to the invention can be carried out in vitro, inthe presence of isolated enzymes (or enzyme systems additionallycomprising one or more cofactors). In vitro preferably means in acell-free system.

In one embodiment, the enzymes employed in the method are used inpurified form to convert 3-hydroxyalkanoates to alkenes. However, such amethod may be costly, since enzyme and substrate production andpurification costs are high.Thus, in another preferred embodiment, the enzymes employed in themethod are present in the reaction as a non-purified extract, or else inthe form of non-lysed bacteria, so as to economize on proteinpurification costs. However, the costs associated with such a method maystill be quite high due to the costs of producing and purifying thesubstrates.

Accordingly, in one preferred embodiment, the enzymes, native orrecombinant, purified or not, are used to convert a 3-hydroxyalkanoateto an alkene. To do this, the enzymes are incubated in the presence ofthe substrate in physicochemical conditions allowing the enzymes to beactive, and the incubation is allowed to proceed for a sufficient periodof time. At the end of the incubation, one optionally measures thepresence of the alkene by using any detection system known to one ofskill in the art such as gas chromatography or colorimetric tests formeasuring the formation of the alkene product, or of free phosphate, orelse for measuring the disappearance of the 3-hydroxyalkanoate substrateor of ATP.

In a preferred embodiment, cofactors are added so as to best mimic thenatural reaction or so as to provide steric or electroniccomplementation in the catalytic cleft. For example, if one of theenzymes used in the method according to the invention is an enzyme whichnaturally uses mevalonate disphosphate (MDP) as a substrate, thestructure of 3-hydroxyalkanoates leaves a large space in the catalyticcleft empty during enzyme-substrate binding since generally a3-hydroxyalkanoate corresponds to a fragment of MDP. Filling this spacewith a cofactor to replace the missing part of the substrate has thepurpose of most closely mimicking the MDP molecule. As the cofactor isnot modified during the reaction, it will therefore be added only incatalytic amounts. In the case where the substrate of the reaction is3-hydroxypropionate, the complementary cofactor will be propyldiphosphate. In the case where the substrate is 3-hydroxybutyrate or3-hydroxy-3-methyl butyrate, the complementary cofactor will be ethyldiphosphate. In the case where the substrate is 3-hydroxyvalerate or3-hydroxy-3-methylvalerate, the complementary cofactor will be methyldiphosphate. These different molecules are shown in FIG. 5. By chance,it may happen that the complementary cofactor of a reaction has apositive effect on the reaction of another substrate. Generally, thecofactor can be any molecule comprising a phosphoanhydride, andtherefore having the general global formula R—PO₂H—O—PO₃H₂, in which Ris in particular H, a linear, branched or cyclic alkyl group, preferablyhaving from 1 to 10 or from 1 to 5 carbon atoms, or any other monovalentorganic group. The analogous motifs corresponding to methylenediphosphonate monoesters, having the general formula R—O—PO₂H—CH₂—PO₃H₂in which phosphyanhydride is replaced by a methylene bridge having theadvantage of not being hydrolyzed, are also part of the invention. Moregenerally, the cofactors can be monophosphate, or even phosphate-free,analogs of the previous molecules, or else any other molecule that canimprove the reaction yield by providing steric or electroniccomplementation in the enzyme catalytic site. The cofactor isadvantageously selected from the group consisting of the pyrophosphateion, methyl diphosphate, ethyl diphosphate, or propyl diphosphate.

In a preferred embodiment, the conversion occurs in the presence of aco-substrate, said co-substrate preferably being a compound containing aphosphoanhydride, and preferably being ATP, an rNTP, a dNTP or a mixtureof several of these molecules, a polyphosphate, or pyrophosphate. Theco-substrate is generally present in the host. However, in anotherparticular embodiment, a co-substrate can be added to the reaction,preferably selected from the group consisting of ATP, an rNTP, a dNTP, amixture of several rNTPs or dNTPs, a polyphosphate, and preferablypyrophosphate, or a compound containing a phosphoanhydride (representedby the general formula X—PO₃H₂ of FIG. 2).

Although the decarboxylation step, i.e. the reaction defined as (ii)herein-above, does not require ATP consumption, it could be shown thatthe presence of ATP in the reaction could be beneficial. This has beendemonstrated in Example 7, using 3-phosphonoxyisovalerate as asubstrate. It is assumed that ATP might have an effect on the folding ofthe protein by the binding of ATP to the ATP-binding site of thediphosphomevalonate decarboxylase. In fact, this can be observed by eye:the purified enzyme has a tendency to precipitate, and the addition ofATP prevents this effect. It is considered that not only ATP but alsoother similar compounds like dATP, ADP, AMP or other NTPs or dNTPs havethis effect. Thus, in a preferred embodiment, the method according tothe present invention is carried with ATP, dATP, ADP, AMP or an NTPother than ATP or a dNTP as co-substrate.

In another preferred embodiment the method according to the invention iscarried out in culture, in the presence of an organism, preferably amicroorganism, producing the enzymes. Thus, in such an embodiment of theinvention, an organism, preferably a microorganism, that produces theenzymes specified in (i) and (ii) above is used. In a preferredembodiment, the (micro)organism is recombinant in that the enzymesspecified in (i) and (ii) produced by the host are heterologous relativeto the production host. The method can thus be carried out directly inthe culture medium, without the need to separate or purify the enzymes.In an especially advantageous manner, a (micro)organism is used havingthe natural or artificial property of endogenously producing one or more3-hydroxyalkanoates, and also expressing or overexpressing the enzymesspecified in (i) and (ii) above, natural or modified, so as to producealkenes directly from a carbon source present in solution.

For example, the method according to the invention can be carried out byusing microorganisms which produce one or more 3-hydroxyalkanoates [forexample Alcaligenes eutrophus or Bacillus megaterium, or else an E. colistrain genetically modified so as to produce said product(s)] and whichhave been genetically engineered such that they overexpress the enzymesas defined in (i) and (ii) above, said enzymes preferably originatingfrom an organism different from the host microorganism. The geneticmodification can consist, e.g. in integrating the corresponding genesencoding the enzymes into the chromosome, expressing the enzymes from aplasmid containing a promoter upstream of the enzyme-coding sequence,the promoter and coding sequence preferably originating from differentorganisms, or any other method known to one of skill in the art.Alternatively, other bacteria or yeasts may have specific advantages andcan be chosen. For instance, a yeast such as Saccharomyces cerevisiae,an extremophilic bacterium such as Thermus thermophilus, or anaerobicbacteria from the family Clostridiae, microalgae, or photosyntheticbacteria can be used.

The organisms used in the invention can be prokaryotes or eukaryotes,preferably, they are microorganisms such as bacteria, yeasts, fungi ormolds, or plant cells or animal cells. In a particular embodiment, themicroorganisms are bacteria, preferably of the genus Escherichia,Alcaligenes or Bacillus and even more preferably of the speciesEscherichia coli, Alcaligenes eutrophus or Bacillus megaterium.

In another preferred embodiment, the microorganisms are recombinantbacteria of the genus Escherichia, preferably of the species Escherichiacoli, having been modified so as to endogenously produce one or more3-hydroxyalkanoates, and converting them to alkenes.

In a further preferred embodiment the microorganism is a fungus, morepreferably a fungus of the genus Saccharomyces, Schizosaccharomyces,Aspergillus or Trichoderma and even more preferably of the speciesSaccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus nigeror of the species Trichoderma reesei. In a particularly preferredembodiment the microorganism is a recombinant yeast producing3-hydroxyalkanoates and converting them to alkenes due to the expressionof the enzymes specified in (i) and (ii) above.

In another preferred embodiment, the method according to the inventionmakes use of a photosynthetic microorganism expressing the enzymes asspecified in (i) and (ii) above. Preferably, the microorganism is aphotosynthetic bacterium, or a microalgae. Even more preferably such amicroorganism has the natural or artificial property of endogenouslyproducing one or more 3-hydroxyalkanoates. In this case themicroorganism would be capable of producing alkenes directly from CO₂present in solution.

It is also conceivable to use in the method according to the inventionone microorganism that produces an enzyme as defined in (i) above andanother microorganism which produces an enzyme as defined in (ii) above.Moreover, in a further embodiment at least one of the microorganisms iscapable of producing one or more 3-hydroxyalkanoates or, in analternative embodiment, a further microorganism is used in the methodwhich is capable of producing one or more 3-hydroxyalkanoates.

In another preferred embodiment the method according to the inventionmakes use of a multicellular organism expressing the enzymes as definedin (i) and (ii) above. Examples for such organisms are plants oranimals.

In a particular embodiment, the method involves culturing microorganismsin standard culture conditions (30-37° C. at 1 atm, in a fermenterallowing aerobic growth of the bacteria) or non-standard conditions(higher temperature to correspond to the culture conditions ofthermophilic organisms, for example).

In a further preferred embodiment the method of the invention is carriedout in microaerophilic conditions. This means that the quantity ofinjected air is limiting so as to minimize residual oxygenconcentrations in the gaseous effluents containing the alkenehydrocarbons.

In another preferred embodiment the method according to the inventionfurthermore comprises the step of collecting gaseous alkenes degassingout of the reaction, i.e. recovering the products which degas, e.g., outof the culture. Thus in a preferred embodiment, the method is carriedout in the presence of a system for collecting alkene under gaseous formduring the reaction.

As a matter of fact, short alkenes, and particularly ethylene, propyleneand butene isomers, adopt the gaseous state at room temperature andatmospheric pressure.

The method according to the invention therefore does not requireextraction of the product from the liquid culture medium, a step whichis always very costly when performed at industrial scale. The evacuationand storage of the gaseous hydrocarbons and their possible subsequentphysical separation and chemical conversion can be performed accordingto any method known to one of skill in the art.

In a particular embodiment, the method also comprises detecting thealkene (for example propylene, ethylene or isobutylene) which is presentin the gaseous phase. The presence of the compound to be produced in anenvironment of air or another gas, even in small amounts, can bedetected by using various techniques and in particular by using gaschromatography systems with infrared or flame ionization detection, orby coupling with mass spectrometry.

In a particular embodiment, the alkenes produced by a method accordingto the invention are condensed, then optionally reduced, by usingtechniques known to one of skill in the art, so as to produce longerchain alkenes, or longer chain alkanes. For example, isobutylene can beused to synthesize isooctane: the catalytic methods for successfullycarrying out this reaction have already been fully described.

In another embodiment, the method according to the invention ischaracterized by the conversion of a carbon source such as glucose, to3-hydroxyalkanoate, followed by the conversion of said3-hydroxyalkanoate into the corresponding alkene. The different steps ofsaid method are outlined in FIG. 6.

In a particular embodiment, the method is characterized by theconversion of polyhydroxyalkanoates into 3-hydroxyalkanoate by using anenzyme or a suitable physicochemical method, followed by the conversionof said 3-hydroxyalkanoate into said alkene. Optionally, thepolyhydroxyalkanoate has been produced by a microorganism or a plantwhose metabolic pathways have been modified to as to produce high yieldsof polyhydroxyalkanoate.

In another embodiment, the method according to the invention comprisesthe production of alkenes from atmospheric CO₂ or from CO₂ artificiallyadded to the culture medium. In this case the method is implemented inan organism which is able to carry out photosynthesis, such as forexample microalgae.

The present invention also relates to a method for producing an alkenecomprising the step of enzymatically converting a 3-phosphonoxyalkanoateinto the corresponding alkene by use of an enzyme which can catalyze theconversion via decarboxylation and dephosphorylation.

As regards the preferred enzyme to be used in such a method, the sameapplies as has been set forth above in connection with (ii) of themethod according to the invention as described herein-above.

Moreover, also with respect to the other preferred embodiments describedabove for the method according to the invention, the same applies to themethod for producing an alkene from a 3-phosphonoxyalkanoate.

The present invention also relates to organisms, preferablymicroorganisms, which produce at least two enzymes, wherein one enzymeis selected from (i) as specified above and the other enzyme is selectedfrom (ii) as specified above. In a preferred embodiment such an organismis a recombinant organism in the sense that it is genetically modifieddue to the introduction of at least one nucleic acid molecule encodingat least one of the above mentioned enzymes. Preferably such a nucleicacid molecule is heterologous with regard to the organism which meansthat it does not naturally occur in said organism.

Thus, the present invention also relates to an organism, preferably amicroorganism, comprising a nucleic acid molecule coding for an enzymeas defined in (i) above and comprising a nucleic acid molecule codingfor an enzyme as defined in (ii) above. In a preferred embodiment atleast one of the nucleic acid molecules is heterologous to the organismwhich means that it does not naturally occur in said organism. Themicroorganism is preferably a bacterium, a yeast or a fungus. In anotherpreferred embodiment the organism is a plant or non-human animal. Asregards other preferred embodiments, the same applies as has been setforth above in connection with the method according to the invention.

Moreover, the present invention also relates to a composition comprisinga microorganism according to the present invention, a suitable culturemedium and a 3-hydroxyalkanoate compound or a carbon source that can beconverted by the microorganism to a 3-hydroxyalkanoate compound.

The present invention also relates to the use of a combination of atleast two enzymes, wherein one enzyme is selected from the following (i)and the other enzyme is selected from the following (ii) or of anorganism, preferably a microorganism, according to the invention or of acomposition according to the invention, for producing alkene compoundsfrom 3-hydroxyalkanoates, wherein (i) and (ii) are as follows:

-   -   (i) a first enzyme having an activity of converting the        3-hydroxyalkanoate into the corresponding        3-phosphonoxyalkanoate; and    -   (ii) a second enzyme being different from the first enzyme and        having an activity of converting said 3-phosphonoxyalkanoate        into said alkene.

As regards the preferred embodiments of the different componentsrecited, the same applies as has been set forth above in connection withthe method according to the invention.

Other aspects and advantages of the invention will be described in thefollowing examples, which are given for purposes of illustration and notby way of limitation.

FIGURES LEGENDS

FIG. 1: The 3-hydroxypropionate motif.

FIG. 2: Reaction catalyzed by mevalonate diphosphate decarboxylase.

FIG. 3: Examples of 3-hydroxyalkanoates.

FIG. 4: Production of alkenes from 3-hydroxyalkanoates by combining twoenzymatic steps.

FIG. 5: Cofactors that can be used in the reaction for the purpose ofstructural complementation in the catalytic site of mevalonatediphosphate decarboxylase.

FIG. 6: Integrated method for producing an alkene from glucose.

FIG. 7: Screening of MDP decarboxylases in a complementation assay. Thereaction catalyzed by the P. torridus (SEQ ID NO:1) enzyme alone (0.1mg) without a second enzyme, was taken as reference.

FIG. 8: Combined effect of MDP decarboxylase enzymes from P. torridus(SEQ ID NO:1) and S. gordonii (SEQ ID NO:5) for converting3-hydroxyisovalerate (HIV) into isobutene (IBN). IBN production wasmeasured as a function of the concentration of S. gordonii MDPdecarboxylase (SEQ ID NO:5) added to a pre-incubated reaction mixture ofHIV with 100 μg of P. torridus MDP decarboxylase (SEQ ID NO:1).

FIG. 9: Screening of enzyme homologs of S. gordonii MDP decarboxylase(SEQ ID NO:5). The peak area of isobutene obtained for the reaction withTh. acidophilum (SEQ ID NO:2) (0.1 mg) enzyme alone (no second enzyme),was used as reference (ratio=1).

-   -   MDP decarboxylases from the Streptococcus genus are particularly        efficient when used in combination with an enzyme of the P.        torridus phylum.

FIG. 10: Scheme of the ADP quantification assay, monitoring NADHconsumption by the decrease of absorbance at 340 nm.

FIG. 11: Plot of the velocity as a function of substrate concentrationfor the phosphotransferase reaction catalyzed by P. torridus MDPdecarboxylase (SEQ ID NO:1). Initial rates were computed from thekinetics over the 30 first minutes of the reaction.

FIG. 12: Isobutene production from 3-hydroxyisovalerate in the followingassays:

-   -   Without enzyme    -   In the presence of S. mitis MDP decarboxylase (SEQ ID NO:10)    -   In the presence of Th. acidophilum MDP decarboxylase (SEQ ID        NO:2)    -   In the presence of both Th. acidophilum (SEQ ID NO:2) and S.        mitis (SEQ ID NO:10) enzymes.

FIG. 13: Scheme for the chemical synthesis of 3-phosphonoxyisovalerate.

FIG. 14: GC analysis of assays for isobutene production from3-phosphonoxyisovalerate in the absence and presence of ATP.

-   -   Assays:    -   1. Without enzyme, 0 mM ATP    -   2. 2 mg/ml enzyme, 0 mM ATP    -   3. Without enzyme, 10 mM ATP    -   4. 2 mg/ml enzyme, 10 mM ATP

The following Examples serve to illustrate the invention.

EXAMPLES Example 1 Cloning, Expression and Purification of an MDPDecarboxylase Library

A library of 55 genes encoding representatives of thediphosphomevalonate decarboxylase (MDP decarboxylase) family acrosseukaryotic, prokaryotic and archaeal organisms was constructed andtested to identify the most active candidates for improving isobutene(IBN) production.

Cloning, Bacterial Cultures and Expression of Proteins.

The genes encoding mevalonate diphosphate (MDP) decarboxylase EC4.1.1.33 were cloned in the pET 25b vector (Novagen) in the case ofeukaryotic genes and in pET 22b (Novagen) in the case of prokaryoticgenes. A stretch of 6 histidine codons was inserted after the methionineinitiation codon to provide an affinity tag for purification. CompetentE. coli BL21(DE3) cells (Novagen) were transformed with these vectorsaccording to the heat shock procedure. The transformed cells were grownwith shaking (160 rpm) on ZYM-5052 auto-induction medium (Studier F W,Prot. Exp. Pur. 41, (2005), 207-234) for 6 h at 37° C. and proteinexpression was continued at 28° C. overnight (approximately 16 h). Thecells were collected by centrifugation at 4° C., 10,000 rpm for 20 minand the pellets were frozen at −80° C.

Protein Purification and Concentration.

The pellets from 200 ml of culture cells were thawed on ice andresuspended in 5 ml of Na₂HPO₄ pH 8 containing 300 mM NaCl, 5 mM MgCl₂and 1 mM DTT. Twenty microliters of lysonase (Novagen) were added. Cellswere incubated 10 minutes at room temperature and then returned to icefor 20 minutes. Cell lysis was completed by sonication for 3×15 seconds.The bacterial extracts were then clarified by centrifugation at 4° C.,10,000 rpm for 20 min. The clarified bacterial lysates were loaded onPROTINO-1000 Ni-TED column (Macherey-Nagel) allowing adsorption of 6-Histagged proteins. Columns were washed and the enzymes of interest wereeluted with 4 ml of 50 mM Na₂HPO₄ pH 8 containing 300 mM NaCl, 5 mMMgCl₂, 1 mM DTT, 250 mM imidazole. Eluates were then concentrated anddesalted on Amicon Ultra-4 10 kDa filter unit (Millipore) andresuspended in 0.25 ml 50 mM Tris-HCl pH 7.4 containing 0.5 mM DTT and 5mM MgCl₂. Protein concentrations were quantified according to theBradford method. The purity of proteins thus purified varied from 40% to90%.

Example 2 Screening of the MDP Decarboxylase Library

MDP decarboxylases were evaluated using a complementation assay. P.torridus MDP decarboxylase (SEQ ID NO:1) was incubated together witheach tested enzyme from the library.

The enzymatic assay was carried out under the following conditions:

50 mM Tris HCl pH 7,0 10 mM MgCl₂ 20 mM KCl 40 mM ATP

50 mM 3-hydroxyisovalerate (HIV)The pH was adjusted to 7.0

100 μg of the MDP decarboxylase from P. torridus (SEQ ID NO:1) and 1 mgof the MDP decarboxylase to be tested were added to 1 ml of reactionmixture. A reaction mixture containing only 100 μg of P. torridus MDPdecarboxylase (SEQ ID NO:1) was used as reference. The mixture was thenincubated without shaking at 45° C. for 90 h in a sealed vial(Interchim).

One ml of the gaseous phase was collected and injected into a HP5890 gaschromatograph (HP) equipped with an FID detector and a CP SilicaPlotcolumn (Varian). Commercial isobutene was used as reference.

This screening procedure led to the identification of several MDPdecarboxylase enzymes increasing the isobutene production rate. As shownin FIG. 7, a higher production of isobutene was observed for thefollowing MDP decarboxylases.

Candidate 1:

Accession number Genbank: CA197800Accession number SwissProt/TrEMBL: Q1GAB2Organism: Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 (SEQ IDNO:9)

Candidate 2:

Accession number Genbank: AAC50440.1Accession number SwissProt/TrEMBL: P53602.1Organism: Homo sapiens (SEQ ID NO:8)

Candidate 3:

Accession number Genbank: ABV09606Accession number SwissProt/TrEMBL: A8AUU9Organism: Streptococcus gordonii str. Challis substr. CH1 (SEQ ID NO:6)

The highest production of isobutene was observed with purified MDPdecarboxylase from Streptococcus gordonii.

This indicated that the two enzymes present in the assay (the one fromP. torridus (SEQ ID NO:1) and the other from S. gordonii (SEQ ID NO:5))were performing complementarily the two steps of reaction producing IBNfrom HIV: transfer of the terminal phosphoryl group from ATP to theC3-oxygen of 3-hydroxyisovalerate followed by combineddephosphorylation-decarboxylation of the intermediate3-phosphonoxyisovalerate.

Example 3 Effect of Enzyme Concentration on Isobutene Production Yield

The effect of Streptococcus gordonii MDP decarboxylase (SEQ ID NO:5)concentration was assessed under the following conditions:

50 mM Tris-HCl pH 7,0 10 mM MgCl₂ 20 mM KCl 40 mM ATP

50 mM 3-hydroxyisovalerate (HIV)The pH was adjusted to 7,0

100 μg of MDP decarboxylase from P. torridus (SEQ ID NO:1) and a varyingamount (from 0 to 1 mg) of purified MDP decarboxylase from Streptococcusgordonii (SEQ ID NO:5) were added to 1 ml of reaction mixture. Themixture was then incubated without shaking at 45° C. for 90 h in asealed vial (Interchim).

One ml of the headspace phase was collected and injected into a HP5890gas chromatograph (HP) equipped with an FID detector and a CP SilicaPlotcolumn (Varian). Commercial isobutene was used as reference.

Increasing the S. gordonii enzyme (SEQ ID NO:5) concentration resultedin an increase of the amount of isobutene produced (FIG. 8).

Example 4 Screening of a Library of Streptococcus gordonii MDPDecarboxylase Homologs

Using the BLAST online program hosted by NCBI, sequences were searchedagainst non redundant protein sequence database to generate a list ofenzymes with high sequence similarity (>40% identity) to theStreptococcus gordonii enzyme (SEQ ID NO:5). The resulting list included18 candidates.

Identity with MDP decarboxylase from Accession Streptococcus numberMicroorganisms gordonii Genbank Streptococcus oralis ATCC 35037 75EFE56694.1 Leptotrichia goodfellowii F0264 61 EEY36155.1 Carnobacteriumsp. AT7 40 EDP67928.1 Enterococcus faecium TX1330 40 EEI60970.1Staphylococcus aureus JH1 40 ABR51487.1 Streptococcus agalactiae NEM31670 CAD47054.1 Streptococcus mutans UA159 71 AAN58642.1 Streptococcusuberis 0140J 71 CAR41735.1 Streptococcus infantarius subsp 71 EDT48420.1infantarius ATCC BAA-102 (SEQ ID NO:7) Streptococcus gallolyticus UCN3471 CBI13757.1 (SEQ ID NO:11) Streptococcus dysgalactiae subsp 71BAH81333.1 equisimilis GGS_124 Streptococcus sp. M143 76 EFA24040.1 (SEQID NO:13) Streptococcus salivarius SK126 74 EEK09252.1 (SEQ ID NO:15)Streptococcus suis 89/1591 40 EEF63672.1 (SEQ ID NO:14) Streptococcusparasanguinis ATCC 73 EFH19018.1 15912 Streptococcus sanguinis SK36 98ABN43791.1 (SEQ ID NO:12) Streptococcus sp. 2_1_36FAA 98 EEY81027.1Streptococcus mitis B6 74 CBJ22986.1 (SEQ ID NO:10)

Sequences of MDP decarboxylase enzymes inferred from the genomes of theabove species as well as from the genome of S. gordonii (SEQ ID NO:5)were generated by oligonucleotide concatenation to fit the codon usageof E. coli. A stretch of 6 histidine codons was inserted after themethionine initiation codon to provide an affinity tag for purification.The genes thus synthesized were cloned in a pET25b expression vector(the vectors were constructed by GENEART AG). After transformation ofthe E. coli strain BL21(DE3), the proteins were produced according tothe protocol described in Example 1. The enzymes were then assayed usingthe method described in Example 2, using Th. acidophilum MDPdecarboxylase (SEQ ID NO:2) instead of the P. torridus enzyme. Thisscreening procedure led to the identification of enzymes more efficientfor isobutene production than the S. gordonii enzyme (SEQ ID NO:5) (FIG.9), in particular MDP decarboxylases from S. infantarius (SEQ ID NO:7),S. gallolyticus (SEQ ID NO:11), S. sp. M143 (SEQ ID NO:13), S.salivarius (SEQ ID NO:15), S. suis (SEQ ID NO:14), S. sanguinis (SEQ IDNO:12) and S. mitis (SEQ ID NO:10).

Example 5 Characterisation of the Phosphotransferase Activity

The release of ADP that is associated with IBN production from HIV wasquantified using the pyruvate kinase/lactate dehydrogenase coupled assay(FIG. 10). The MDP decarboxylases from P. torridus (SEQ ID NO:1), Th.Acidophilum (SEQ ID NO:2), S. infantarius (SEQ ID NO:7), S. mitis (SEQID NO:10) were evaluated for their ability to phoshorylate HIV,releasing ADP.

The studied enzymatic reaction was carried out under the followingconditions at 40° C.:

50 mM Tris-HCl pH 7,0 10 mM MgCl₂ 100 mM KCl 5 mM ATP 0.2 mM NADH 0.5 mMPhosphoenolpyruvate

3 U/ml Lactate dehydrogenase1.5 U/ml Pyruvate kinase

0-50 mM 3-Hydroxyisovalerate (HIV)

The pH was adjusted to 7,0.

Each assay was started by addition of particular enzyme (at aconcentration from 0.05 to 1 mg/ml) and the disappearance of NADH wasmonitored by following the absorbance at 340 nM.

Assays with MDP decarboxylases from the P. torridus phylum as well fromthe Streptococcus genus gave rise to a reproducible increase in ADPproduction in the presence of HIV. FIG. 11 shows an example of aMichaelis-Menten plot corresponding to the data collected for P.torridus enzyme. The kinetic parameters are shown in the followingTable.

The enzymes from the P. torridus phylum displayed higherphosphotransferase activities than those of the Streptococcus genus.

Example 6 Isobutene Production from 3-Hydroxyisovalerate by CombiningTwo Enzymes

The desired enzymatic reaction was carried out under the followingconditions:

50 mM Tris HCl pH 7,5 10 mM MgCl₂ 20 mM KCl 40 mM ATP 50 mM HIV

The pH was adjusted to 7,5

100 μg of MDP decarboxylase from Th. acidophilum (SEQ ID NO:2) and 500μg of MDP decarboxylase from S. mitis (SEQ ID NO:10) were added to 1 mlof reaction mixture. Control reactions with only one of the two enzymeswere run in parallel. The assays were incubated without shaking at 37°C. in a sealed vial (Interchim).

The production of IBN was measured by analyzing aliquots sampled over a142 hour incubation period.

One ml of the gaseous phase was collected and injected into a HP5890 gaschromatograph (HP) equipped with an FID detector and a CP SilicaPlotcolumn (Varian). Commercial isobutene was used as reference.

The kinetics of isobutene production is shown in FIG. 12. MDPdecarboxylase from Th. acidophilum (SEQ ID NO:2) catalyzed theproduction of isobutene from HIV. The addition of MDP decarboxylase fromS. mitis (SEQ ID NO:10) led to a 3-fold increase of isobutene productionafter 142 h of incubation.

MDP decarboxylase from S. mitis (SEQ ID NO:10) alone produced only smallamounts of isobutene after 6 days of incubation, indicating a lowphosphotransferase activity.

k_(cat)/ KM × 10⁻³, Organism K_(M), mM k_(cat), sec⁻¹ mM⁻¹ sec⁻¹Thermoplasma acidophilum 4.02 0.26 60 (SEQ ID NO:2) Picrophilus torridus9.17 0.19 20 (SEQ ID NO:1) Streptococcus mitis 12.1 0.04 3 (SEQ IDNO:10) Streptococcus infantarius 13.4 0.03 2 (SEQ ID NO:7)

Isobutene production can thus be increased by combining two types ofenzymes performing complementarily the two reaction steps.

Example 7 Effect of ATP on Isobutene Production from3-Phosphonoxyisovalerate (PIV)

The compound 3-phosphonoxyisovalerate (PIV) was chemically synthesizedfrom 3-hydroxyisovalerate according to the scheme depicted in FIG. 13 bySYNTHEVAL (France).

The assays of isobutene production were carried out under the followingconditions:

50 mM Tris-HCl pH 7,5 10 mM MgCl₂ 20 mM KCl

0 mM ATP (assay N21 and N22)10 mM ATP (assay N23 and N24)25 mM 3-phosphonoxyisovalerateThe pH was adjusted to 7.5

The reaction was initiated by addition of 2 mg of purified MDPdecarboxylase from S. mitis (SEQ ID NO:10) to 0.5 ml of reactionmixture. Control reactions were run in the absence of enzyme (assays No1 and No 3).

The mixture was incubated without shaking at 37° C. for 26 h in a sealedvial of 2 ml (Interchim).

One ml of the gaseous phase was collected and injected into a Varian430-GC gas chromatograph equipped with an FID detector and a CPSilicaPlot column (Varian). Commercial isobutene was used as reference.

Addition of 10 mM ATP to the reaction mixture increased 120 foldisobutene production from 3-phosphonoxyisovalerate (PIV) (FIG. 14).

Example 8 Kinetic Parameters of Isobutene Production from3-Phosphonoxyisovalerate (PIV)

The kinetic parameters of isobutene production were measured under thefollowing conditions:

50 mM Tris-HCl pH 7,5 10 mM MgCl₂ 50 mM KCl 40 mM ATP

0-100 mM 3-phosphonoxyisovalerateThe pH was adjusted to 7,5

The reaction was initiated by addition of 1 mg of purified MDPdecarboxylase from S. mitis (SEQ ID NO:10) to 0.5 ml of reactionmixture. The mixture was then incubated without shaking at 37° C. for 44h in a sealed vial of 2 ml (Interchim).

One ml of the gaseous phase was collected and injected into a Varian430-GC gas chromatograph equipped with an FID detector and a CPSilicaPlot column (Varian). Commercial isobutene was used as reference.

The assays with MDP decarboxylase from S. mitis (SEQ ID NO:10) showed a160-400 fold increase in IBN production over the background level(spontaneous decomposition of 3-phosphonoxyisovalerate) in the presenceof ATP as co-factor (see the following Table).

PIV concentration, Peak area, arbitrary units mM No enzyme 2 mg/mlenzyme 25 164.9 26945.2 50 328.7 65720.4 75 561.5 239249.2 100 2078.7339363.7

MDP decarboxylase from S. mitis (SEQ ID NO:10) was found to have a K_(M)higher than 60 mM and a k_(cat) of at least 1.3×10⁻³ sec-1.

1-19. (canceled)
 20. A method for producing an alkene comprising theconversion of a 3-hydroxyalkanoate into said alkene by: (i) a firstenzyme having an activity of converting the 3-hydroxyalkanoate into thecorresponding 3-phosphonoxyalkanoate; and (ii) a second enzyme beingdifferent from the first enzyme and having an activity of convertingsaid 3-phosphonoxyalkanoate into said alkene.
 21. The method of claim20, wherein the first enzyme is a mevalonate diphosphate (MDP)decarboxylase and the second enzyme is a different mevalonatediphosphate (MDP) decarboxylase.
 22. The method of claim 20 wherein: (i)the first enzyme is selected from the group consisting of: (A) a proteincomprising the amino acid sequence as shown in SEQ ID NO: 1 or a proteincomprising an amino acid sequence which is at least 75% identical to theamino acid sequence shown in SEQ ID NO: 1 and showing an activity ofconverting the 3-hydroxyalkanoate into the corresponding3-phosphonoxyalkanoate which is at least as high as the correspondingactivity of the protein having the amino acid sequence shown in SEQ IDNO: 1; (B) a protein comprising the amino acid sequence as shown in SEQID NO: 2 or a protein comprising an amino acid sequence which is atleast 75% identical to the amino acid sequence shown in SEQ ID NO: 2 andshowing an activity of converting the 3-hydroxyalkanoate into thecorresponding 3-phosphonoxyalkanoate which is at least as high as thecorresponding activity of the protein having the amino acid sequenceshown in SEQ ID NO: 2; (C) a protein comprising the amino acid sequenceas shown in SEQ ID NO: 3 or a protein comprising an amino acid sequencewhich is at least 75% identical to the amino acid sequence shown in SEQID NO: 3 and showing an activity of converting the 3-hydroxyalkanoateinto the corresponding 3-phosphonoxyalkanoate which is at least as highas the corresponding activity of the protein having the amino acidsequence shown in SEQ ID NO: 3; and (D) a protein comprising the aminoacid sequence as shown in SEQ ID NO: 4 or a protein comprising an aminoacid sequence which is at least 75% identical to the amino acid sequenceshown in SEQ ID NO: 4 and showing an activity of converting the3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate whichis at least as high as the corresponding activity of the protein havingthe amino acid sequence shown in SEQ ID NO: 4; and (ii) the secondenzyme is selected from the group consisting of: (a) a proteincomprising the amino acid sequence as shown in SEQ ID NO: 5 or a proteincomprising an amino acid sequence which is at least 75% identical to theamino acid sequence shown in SEQ ID NO: 5 and showing an activity ofconverting said 3-phosphonoxyalkanoate into said alkene which is atleast as high as the corresponding activity of the protein having theamino acid sequence shown in SEQ ID NO: 5; (b) a protein comprising theamino acid sequence as shown in SEQ ID NO: 6 or a protein comprising anamino acid sequence which is at least 75% identical to the amino acidsequence shown in SEQ ID NO: 6 and showing an activity of convertingsaid 3-phosphonoxyalkanoate into said alkene which is at least as highas the corresponding activity of the protein having the amino acidsequence shown in SEQ ID NO: 6; (c) a protein comprising the amino acidsequence as shown in SEQ ID NO: 7 or a protein comprising an amino acidsequence which is at least 75% identical to the amino acid sequenceshown in SEQ ID NO: 7 and showing an activity of converting said3-phosphonoxyalkanoate into said alkene which is at least as high as thecorresponding activity of the protein having the amino acid sequenceshown in SEQ ID NO: 7; (d) a protein comprising the amino acid sequenceas shown in SEQ ID NO: 8 or a protein comprising an amino acid sequencewhich is at least 75% identical to the amino acid sequence shown in SEQID NO: 8 and showing an activity of converting said3-phosphonoxyalkanoate into said alkene which is at least as high as thecorresponding activity of the protein having the amino acid sequenceshown in SEQ ID NO: 8; (e) a protein comprising the amino acid sequenceas shown in SEQ ID NO: 9 or a protein comprising an amino acid sequencewhich is at least 75% identical to the amino acid sequence shown in SEQID NO: 9 and showing an activity of converting said3-phosphonoxyalkanoate into said alkene which is at least as high as thecorresponding activity of the protein having the amino acid sequenceshown in SEQ ID NO: 9 (f) a protein comprising the amino acid sequenceas shown in SEQ ID NO: 10 or a protein comprising an amino acid sequencewhich is at least 75% identical to the amino acid sequence shown in SEQID NO: 10 and showing an activity of converting said3-phosphonoxyalkanoate into said alkene which is at least as high as thecorresponding activity of the protein having the amino acid sequenceshown in SEQ ID NO: 10; (g) a protein comprising the amino acid sequenceas shown in SEQ ID NO: 11 or a protein comprising an amino acid sequencewhich is at least 75% identical to the amino acid sequence shown in SEQID NO: 11 and showing an activity of converting said3-phosphonoxyalkanoate into said alkene which is at least as high as thecorresponding activity of the protein having the amino acid sequenceshown in SEQ ID NO: 11; (h) a protein comprising the amino acid sequenceas shown in SEQ ID NO: 12 or a protein comprising an amino acid sequencewhich is at least 75% identical to the amino acid sequence shown in SEQID NO: 12 and showing an activity of converting said3-phosphonoxyalkanoate into said alkene which is at least as high as thecorresponding activity of the protein having the amino acid sequenceshown in SEQ ID NO: 12; (i) a protein comprising the amino acid sequenceas shown in SEQ ID NO: 13 or a protein comprising an amino acid sequencewhich is at least 75% identical to the amino acid sequence shown in SEQID NO: 13 and showing an activity of converting said3-phosphonoxyalkanoate into said alkene which is at least as high as thecorresponding activity of the protein having the amino acid sequenceshown in SEQ ID NO: 13; (j) a protein comprising the amino acid sequenceas shown in SEQ ID NO: 14 or a protein comprising an amino acid sequencewhich is at least 75% identical to the amino acid sequence shown in SEQID NO: 14 and showing an activity of converting said3-phosphonoxyalkanoate into said alkene which is at least as high as thecorresponding activity of the protein having the amino acid sequenceshown in SEQ ID NO: 14; and (k) a protein comprising the amino acidsequence as shown in SEQ ID NO: 15 or a protein comprising an amino acidsequence which is at least 75% identical to the amino acid sequenceshown in SEQ ID NO: 15 and showing an activity of converting said3-phosphonoxyalkanoate into said alkene which is at least as high as thecorresponding activity of the protein having the amino acid sequenceshown in SEQ ID NO:
 15. 23. The method of claim 22, wherein: (a) thefirst enzyme is a protein comprising the amino acid sequence as shown inSEQ ID NO: 2 or a protein comprising an amino acid sequence which is atleast 75% identical to the amino acid sequence shown in SEQ ID NO: 2 andshowing an activity of converting the 3-hydroxyalkanoate into thecorresponding 3-phosphonoxyalkanoate which is at least as high as thecorresponding activity of the protein having the amino acid sequenceshown in SEQ ID NO: 2; and (b) the second enzyme is a protein comprisingthe amino acid sequence as shown in SEQ ID NO: 10 or a proteincomprising an amino acid sequence which is at least 75% identical to theamino acid sequence shown in SEQ ID NO: 10 and showing an activity ofconverting said 3-phosphonoxyalkanoate into said alkene which is atleast as high as the corresponding activity of the protein having theamino acid sequence shown in SEQ ID NO:
 10. 24. The method of claim 20,wherein the 3-hydroxyalkanoate is 3-hydroxypropionate and the alkene isethylene.
 25. The method of claim 20, wherein the 3-hydroxyalkanoate is3-hydroxybutyrate and the alkene is propylene.
 26. The method of claim20, wherein the 3-hydroxyalkanoate is 3-hydroxyvalerate and the alkeneis 1-butylene.
 27. The method of claim 20, wherein the hydroxyalkanoateis 3-hydroxy-3-methylbutyrate and the alkene is isobutylene.
 28. Themethod of claim 20, wherein the 3-hydroxyalkanoate is3-hydroxy-3-methylvalerate and the alkene is isoamylene.
 29. The methodof claim 20, characterized in that the conversion step is carried out invitro, in a cell-free system.
 30. The method of claim 20 characterizedin that the method is carried out in the presence of a microorganismproducing said enzymes as defined in (i) and (ii) of claim
 20. 31. Themethod of claim 20, characterized by the use of a multicellular organismproducing said enzymes as defined in (i) and (ii) of claim
 20. 32. Themethod of claim 20, comprising a step of collecting gaseous alkenesdegassing out of the reaction.
 33. A multicellular organism or amicroorganism which produces at least two enzymes, wherein one enzyme isselected from (i) and the other enzyme is selected from (ii), wherein(i) and (ii) are as follows: (i) a first enzyme having an activity ofconverting the 3-hydroxyalkanoate into the corresponding3-phosphonoxyalkanoate; and (ii) a second enzyme being different fromthe first enzyme and having an activity of converting said3-phosphonoxyalkanoate into said alkene.
 34. A method of using themulticellular organism or microorganism of claim 33 to produce alkenes,comprising the steps of: (i) culturing the multicellular organism ormicroorganism of claim 33 with 3-hydroxyalkanoate for a sufficientperiod of time to allow for the conversion of a 3-hydroxyalkanoate to analkene; and (ii) recovering said alkene.
 35. A composition comprisingthe microorganism of claim 33, a suitable culture medium and a3-hydroxyalkanoate compound or a carbon source that can be converted bythe microorganism to a 3-hydroxyalkanoate compound.
 36. A method ofusing a combination of at least two enzymes to convert a3-hydroxyalkanoate to an alkene, wherein the method comprises: (i)contacting a 3-hydroxyalkanoate with: (a) a first enzyme having anactivity of converting the 3-hydroxyalkanoate into the corresponding3-phosphonoxyalkanoate; and (b) a second enzyme being different from thefirst enzyme and having an activity of converting said3-phosphonoxyalkanoate into said alkene; (ii) converting said3-hydroxyalkanoate to an alkene.
 37. A method for producing an alkenecomprising the step of enzymatically converting a 3-phosphonoxyalkanoateinto the corresponding alkene by use of an enzyme which can catalyze theconversion via decarboxylation and dephosphorylation.
 38. The method ofclaim 20, wherein the method is carried out with ATP, dATP, ADP, AMP, anNTP other than ATP, a dNTP or pyrophosphate as co-substrate.